Modified oxidic nano-particle with hydrophobic inclusions, method for the production and use of said particle

ABSTRACT

The invention concerns processes for producing modified oxidic nanoparticles with hydrophobic inclusions, in particular metal oxide particles which contain halogen-containing target molecules; the particles produced in this manner and the use thereof especially as a toner, sunscreen agent, insecticide or for labelling biomolecules.

The invention concerns processes for the production of modifiedmetal-oxidic nano-particles with hydrophobic inclusions, in particularmetal oxide particles which contain halogen-containing target molecules;the particles produced in this manner and the use thereof especially asa toner, sunscreen agent, insecticide or for labelling biomolecules.

Latex particles are hydrophobic and are very suitable as a host forhydrophobic molecules and used as such (Kawaguchi, H., Prog. Polym. Sci.25 (2000) 1171-1210). Although organic nanoparticles dispersed in waterare being used increasingly in pharmaceuticals, cosmetics, plantprotection and foods, solvent residues are for example still presentwhich can have an adverse effect on the respective applications. Theseproblems are at present being intensively researched (Horn, D., andRieger, J., Angew. Chem. 113 (2001) 4460-4492).

Since the metal oxide particles are synthesized by wet chemical methodsin a water-ethanol mixture they naturally contain no interferingsurfactants, stabilizers etc. In addition a multifunctional surface ispresent which can be modified depending on the requirements, for examplewith carboxyl functionalities (as biolinkers) or fluoroorganyl groups(to influence the physicochemical surface properties). Metal oxideparticles are by nature hydrophilic and are therefore unsuitable as ahost for hydrophobic molecules.

Nanoparticles based on silicate which are stained with hydrophilic dyeshave been known for a long time in the prior art. They are used in theform of pigments for example as dyes for toners and inks, for plasticmaterials and also as labelling and carrier materials in the medicalengineering field.

On an industrial scale silicate particles are usually produced by flamehydrolysis (e.g. Aerosil®). Silicate particles obtained in this mannercan be coloured on their surface or in layers.

U.S. Pat. No. 5,102,763 describes the use of hydrophilic, coloured SiO₂particles for use as toners. The surface of these particles iscovalently stained by reacting pre-activated silicate particles withvarious dyes.

The production of coloured particles by covalently binding a dye to thesurface of particles is described in WO 93/10190.

Silicate particles which are only coloured on the surface have atendency to lose colour by bleeding. This results in a reduction in thecolour intensity and these particles are also often no longer uniformlycoloured. The use of these particles to produce conjugates that aresuitable for diagnostic agents is not described.

A process for producing coloured particles with a silicate surface isdescribed in the U.S. Pat. No. 5,209,998. The production process isbased on the coating of coloured pigments with a silicate shell. Henceonly the nucleus of these particles is coloured. The use of theparticles in electrostatic toners, plastic materials and inks isdescribed as the application; a diagnostic application is not disclosed.

A process for producing monodisperse silicate particles i.e. silicateparticles of a uniform size, is the sol-gel process. It was firstdescribed by Stöber et al., (Colloid J. Interface Sci. 26 (1968) 62-69).The production of so-called Stöber particles and their properties weresubsequently extensively examined by numerous groups. These studiesencompassed the determination of the synthesis conditions required toobtain certain particle sizes (Van Helden, et al., Colloid J. InterfaceSci. 81 (1981) 354-68; Giesche, H., J. European Ceramic Soc. 14 (1994)189-204, Van Blaaderen, A., and Vrij, A., Adv. Chem. Ser. 234 (1994)83-111) as well as investigations on particle growth and chemicalcomposition (Byers, C. H., et al., Ind. Eng. Chem. Res. 26 (1987)1916-1923; Matsoukas, T., and Goulari, E., Colloid J. Interface Sci. 124(1988) 252-261; Harris, T., et al., J. Non-Cryst. Solids 121 (1990)307-403; Matsoukas, T., and Gulari, E., Colloid J. Interface Sci. 132(1989) 13-21; Badley, R. D., et al., Langmuir 6 (1990) 792-801).

Various methods have been described in the prior art for doping silicateparticles from the sol-gel process with dyes.

Van Blaadereno, et al., Langmuir 8 (1992) 2921-2931 and Quellet, et al.,Colloid J. Interf. Sci. 159 (1993) 150-7 produced Stöber particles thatwere stained with fluorescein isothiocyanate or rhodamine isothiocyanate(Verhaegh and Van Blaaderen, A., Langmuir 10 (1994) 1427-1438). The dyeswere previously reacted with 3-aminopropyltriethoxysilane (AMEO). Inthis case the dye was covalently attached to the surface or covalentlyincorporated into the particles in layers. The resulting inhomogeneousstaining was of secondary importance in these investigations and themethod usually resulted in relatively large particles in a size range ofabout 500 nm diameter. The particles obtained were used as model systemsfor basic research. The large particle size makes silicate particlesproduced in this manner less suitable for diagnostic applications.

Shibata, S., et al., J. Sol-Gel Sci. And Techn. 10 (1997) 263-268physically doped Stöber particles with various hydrophilic dyes such asrhodamine 6G, water-soluble porphyrins, Nile-blue etc. Schwert, R.,Dissertation Würzburg 2000 found that only cationic but not anionic orhydrophobic dyes can be incorporated physically (non-covalently) in theStöber process.

Matijevic, et al., Dyes and Pigments 17 (1991) 323-340 presented Stöberparticles whose surface was modified with 3-aminopropyltriethoxysilaneswhich were linked via the amino group with dyes in a complicatedprocess. The surface of Stöber particles was also modified in variousother manners. These include reactions with3-methacryloxypropyltrimethoxysilane (MEMO), octadecyltrimethoxysilane(ODS) and 3-aminopropyltriethoxysilane (AMEO) (Giesche, H., andMatijevic, E., Dyes and Pigments 17 (1991) 323-340; Van Blaaderen, A.,and Vrij, A., Golloid J. Interface Sci. 156 (1993) 1-18; Badley, R. D.,et al., Langmuir 6 (1990) 792-801; Philipse, A. P., and Vrij, A.,Colloid J. Interface Sci. 12 (1989) 121-136; Van Helden, A. K., andVrij, A., Colloid J. Interface Sci. 81 (1981) 354-368).

Homogeneously coloured silicate particles can be produced in the sol-gelprocess by covalent dye incorporation (EP 1 036 763). However, the dyeshave to be firstly silanized before they can be used in this process. Acovalent incorporation is only possible in this manner.

Many important target molecules for incorporation into nanoparticles andin particular many dyes carry halogen groups as substituents. These dyesare not only hydrophobic but also oleophobic.

Fluorine-containing coatings based on SiO₂ are known (Lotus-Effect, Easyto clean surfaces, adjustment of refractive numbers—Kron J., et al.,2^(nd) Wörlitzer Workshop: Functional layers—adhesive and antiadhesivesurfaces (“Fördergemeinschaft Dünne Schichten e.V.”), Conference paper2000. However, due to the rapid gelling during the particle productionwith fluoroalkyltrialkoxysilanes, no fluorine-containing silicateparticles have yet been synthesized. But these would be desirable inorder to also enclose hydrophobic and especially oleophobic molecules inSiO₂ particles.

Hence the object of the invention was to modify the production processfor metal oxide particles in such a manner that hydrophobic complexes orhydrophobic organic dyes can for example be integrated into SiO₂particles.

Hence the object was to develop a process which enables theincorporation of hydrophobic and in particular oleophobic dyes intometal oxide particles.

The object is achieved by the invention which is defined in more detailin the independent claims. The dependent claims represent preferredembodiments.

It was surprisingly found that it is possible to produce metal oxideparticles in the sol-gel process in the presence offluoroorganylalkoxysilane or arylalkoxysilane and to non-covalentlyincorporate hydrophobic and in particular oleophobic target moleculesinto these nanoparticles in this production process.

The invention concerns sol-gel processes for producing a metal oxideparticle which contains at least one target molecule containing halogenin which, starting from known metal oxide precursors, the said precursorand the said target molecule are used, characterized in that apolyhalogenated metal alkyl-alkoxy compound, in particularalkylalkoxysilane is additionally used in the said sol-gel process.

A sol-gel process is understood as any process which can be used inanalogy to the process described by Stöber et al. (1968), supra toproduce colloidal nanoparticles. The products of this process arereferred to as Stöber particles or nanoparticles.

The invention concerns the non-covalent incorporation ofhalogen-containing target molecules into metal oxide particles. Thetarget molecules in the sense of this invention consist of 5-65 percentby weight (=weight %) halogen and preferably have a molecular weight ofbetween 250 and 5000 Dalton. Target molecules are in particularhalogen-containing dyes and halogen-containing insecticides.

The halogen-containing target molecule is not silanized. Hence theirincorporation into the Stöber particles is non-covalent.

The process according to the invention is especially characterized inthat for the first time it has been possible to produce Stöber particlesin the presence of a polyhalogenated metal alkylalkoxy compound. Theprocess can be carried out in the presence or absence of a targetmolecule. A polyhalogenated metal alkylalkoxy compound contains a linearor branched alkyl residue with 2 to 20 carbon atoms which carries atleast two halogen groups. The polyhalogenated alkyl residue preferablycontains less than 30 halogen groups. Particularly preferredpolyhalogenated metal alkylalkoxy compounds contain alkyl residues with3 to 20 carbon atoms and 2 to 15 halogen groups. Metal alkylalkoxycompounds based on silicon, titanium or zirconium and in particular thealkylalkoxysilane are particularly preferred.

Metal alkoxides or metal halogenides are usually used as metal oxideprecursors. Preferred metal alkoxides are silicon metal oxides inparticular tetraethoxysilane (TEOS) and tetramethoxysilane (TMES).

In the original Stöber process using SiO₂ as the metal oxide, the SiO₂particles are produced by hydrolysis and condensation of a siliconalkoxide which is usually tetraethoxysilane (TEOS). The reaction takesplace in a mixture of water, ammonia and a lower alcohol, usual ethanol.The main reactions in the formation of the SiO₂ particles can bedescribed as follows:

1) Hydrolysis≡Si—OR+H₂O→≡Si—OH+ROH

2) Condensation I≡Si—OR+=≡Si—OH→≡Si—O—Si≡+ROH

3) Condensation II≡Si—OH+≡Si—OH→≡Si—O—Si≡+H₂O

4) Total reactionSi(OR)₄+2H₂O→SiO₂+4 ROH

In the synthesis alcohol, water and ammonia are added first andsubsequently TEOS is added. Depending on the synthesis conditions, thesolution becomes opalescent after a few seconds to minutes. Thisinduction period increases with decreasing particle size andtemperature. The size of the particles obtained has a standard deviationof 2-8%. During the reaction the alcohol serves as a cosolvent for thewater-insoluble TEOS. The ammonia catalyses the hydrolysis as well asthe condensation reaction. The base deprotonates the surface silanolgroups of the formed particles. The resulting negative charges stabilizethe colloidal system as a result of electrostatic repulsion. Hence thesuspensions remain stable for several months to years. At the same timethe silanol groups that are present enable a functionalization of theparticle surface (various examples thereof have already been describedin the literature e.g. AMEO, ETEO, MEMO, MPTMO, GLYMO, GF20. Theirdispersibility in various solvents can be varied by suitable surfacemodification.

The particle size can be controlled by the ammonia and waterconcentration, the reaction temperature and the solvent. The followingtrends are seen:

1) Increasing the ammonia as well as the water concentration acceleratesthe reaction and increases the particle size.

2) The particle size increases and the monodispersity decreases withincreasing chain length and branching of the alcohol.

3) The particle size decreases with an increasing length of the alkoxideresidues of the silane. The ionic strength of the reaction solution(salt effect) can also influence the particle size due to compression ofthe electrostatic double layer.

4) If the TEOS concentration exceeds 0.2 mol/l, the particles becomemore polydisperse and less spherical.

The previously discussed influences on particle formation in theoriginal Stöber process apply analogously to a process according to theinvention in which a polyhalogenated metal alkylalkoxy compound isadditionally used in order to for example incorporate ahalogen-containing target molecule in the Stöber particles obtained bythis process.

The process according to the invention can for example be carried out bysimultaneously reacting the metal oxide precursor, halogen-containingdye and polyhalogenated metal alkylalkoxy compound components undersuitable reaction conditions known to a person skilled in the art.

The halogen-containing target molecule and the polyhalogenated metalalkylalkoxy compound are preferably dissolved in advance in a suitablesolvent, mixed and added together.

A sol-gel process comprising the following steps is particularlypreferred for producing a metal oxide particle containing at least onehalogen-containing target molecule a) production of a mixture containingthe target molecule and a polyhalogenated metal alkylalkoxy compound, b)starting the sol-gel process with a metal oxide precursor, c) adding thesolution from a), d) optionally further addition of the metal oxideprecursor and e) ending the sol-gel process.

The quantity ratios of the metal oxide precursor that are used in theabove steps b) and d) can vary over a wide range. Preferably between 90to 10% of the total amount of metal oxide precursor used in the processis used in step b) and correspondingly the remaining 10 to 90% is usedin step d). The partial amount used in step b) is particularlypreferably 75 to 25% and in step d) 25 to 75%.

Also the time period for starting the sol-gel process in step b) isvariable. It is preferably less than 1 h, more preferably between 1 and20 min and particularly preferably between 2 and 10 min.

It has proven to be particularly suitable to coordinate the molar ratiosof metal oxide precursor and polyhalogenated metal alkylalkoxy compound.Preferably 0.04 to 0.4 mol % polyhalogenated metal alkylalkoxy compound,particularly preferably 0.1 to 0.3 mol % based on the metal oxideprecursor are used.

The halogen-containing target molecules preferably contain between 10and 65 weight, particularly preferably between 15 and 50 weight %halogen and the molecular weight is preferably between 250 and 5000Dalton, more preferably between 300 and 4000 Dalton and especiallypreferably between 400 and 3000 Dalton.

Preferred halogens in the halogen-containing target molecules arefluorine and chlorine.

The amount of added target molecule can vary according to needs. Ofcourse it is also possible to prepare particles which contain no targetmolecules or only minimal amounts thereof. Preferably between 0.1 and10% by weight target molecule and particularly preferably between 0.2and 5 weight % based on the metal oxide precursor is used.

Oxides of the elements from groups III, IV and IVb of the periodicsystem come into special consideration as metal oxides or as componentsof mixed oxides. The metal oxide precursor is preferably selected suchthat in addition to the inclusions of the target molecule and thecovalently incorporated polyhalogenated metal alkyl, the Stöberparticles are essentially composed of B₂O₃, Al₂O₃, SiO₂, SnO₂, ZrO₂ orTiO₂.

Of course particles based on mixed oxides can be used in an analogousmanner in the inventive sol-gel process.

Metal oxide precursors based on boron, silicon or zirconium areparticularly preferably used, silicon precursors being especiallypreferred.

The present invention also concerns the particles that can be obtainedby the process according to the invention.

These are in particular particles which were obtained by hydrolysis andcondensation of sol-gel precursors of elements of groups III, IV, IVb,preferably Si (Ti, Zr, Al) in combination with hydrophobic sol-gelprecursors such as perfluorinated alkyltrialkoxysilanes (e.g.3,3,3-trifluoropropyltrimethoxysilane) orbis(trialkoxysilylalkyl)benzenes (e.g.bis(trimethoxysilylethyl)benzene). These particles preferably containthe above-mentioned target molecules.

Due to the hydrophobic (fluorinated) environment that is present in thecase of the metal oxide particles produced according to the invention,fluorophores for example do not exhibit the otherwise common adverseeffects of water i.e. quenching due to water does not occur.

The modification of the particle interior according to the inventionenables other hydrophobic (e.g. LC Red 640) as well as oleophobicmolecule/complexes to be incorporated into the originally highly polaroxidic matrix in addition to lanthanoid complexes. The particle surfacecan be functionalized as required for example with carboxyl, amino,mercapto, epoxy and aldehyde groups. This can be accomplished amongothers by silanization. The particle size can be adjusted from the nano-to micrometer range with a narrow size distribution.

The particle type (cf. FIG. 1) is not decisive. The particles arepreferably composed of an inorganic-oxidic core. This core can have ahomogeneous (type 1) or heterogeneous (core-shell type (2) or currantcake model (3)) composition.

The Stöber particles according to the invention loaded with ahalogen-containing target molecule can be very advantageously used invarious technical fields. They are especially suitable as labels forbiomolecules and hence for applications of the labelled biomolecules inimmunological and other detection methods, as toners in the printingindustry, as sunscreen agents and as insecticides. It is also possibleto incorporate them into any polymer matrix (e.g. Ormocer®). Theapplications as labels for biomolecules or as an insecticide areparticularly preferred.

The metal oxide particles according to the invention can be subsequentlymodified. Thus the particles can for example be coated with one or moreadditional, preferably colourless layers in order to chemically protectthe particles. The purpose of this coating is to obtain a metal oxidesurface e.g. a silicate-like surface that is as uniform as possible fromwhich colour molecules no longer protrude. This facilitates additionalcoupling with functional groups and biomolecules and reduces the risk ofsecondary reactions with dye molecules on the surface. Preferably anadditional uncoloured silicate layer is applied at a thickness of 1 to30 nm, preferably 2 to 20 nm to the homogeneously coloured silicateparticles.

The metal oxide particles according to the invention can either beprovided directly with functional groups or they can be provided on thesurface of the additional coating layer in order to couple additionalmolecules to the particle which according to the invention arepreferably biomolecules.

The functional groups can in turn be attached to the particles viaspacer or linker molecules. It is important that the functional group tobe introduced is anchored in the network of the metal oxide particle inorder to ensure a stable linkage.

Preferred modification groups are functional groups such as carboxylgroups, amino groups, epoxy groups, hydroxyl groups or thiol groups. Aperson skilled in the art knows how to introduce such groups. It doesnot therefore have to be separately elucidated.

It is preferable to introduce carboxyl groups which is preferablycarried out by reacting the coloured metal oxide particles with a dyeacid anhydride which contains the said silanol group for anchoring inthe particle. In order to activate the functional groups they can forexample be converted into active esters with N-hydroxy-succinimidebefore reaction with the biomolecules to be coupled. All these steps arefamiliar to a person skilled in the art.

The conjugates according to the invention are composed of metal oxideparticles loaded with a halogen-containing dye and biomolecules. Thebiomolecules are preferably coupled via the functional groups that areintroduced on the surface. In general the biomolecules are linked to thesurface of the particle via free amino or carboxyl groups or thiolgroups such that the covalent linkage is preferably via amide orthioether bonds.

Biomolecules in the sense of the present invention are understood as allmolecules that can be used to determine an analyte in a sample, inparticular for an immunological determination of an analyte. The termbiomolecule for example includes proteins, glycoproteins, peptides,nucleic acids, peptidic nucleic acids, saccharides, hormones, haptens,vitamins, naturally occurring or artificially produced binding partnersand antigens. Antibodies and fragments thereof are preferably used asbiomolecules in the conjugate according to the invention. Antibodies areunderstood to include monoclonal as well as polyclonal antibodies andchimeric antibodies and fragments thereof such as Fab, Fc, Fab′,F(ab′)₂, Fv, scfv. Coupling to the biomolecules streptavidin or avidinor biotin is also one of the preferred embodiments of the invention.

Conjugates of the inventive metal oxide particles and biomolecules are afurther subject matter of the invention. These inventive conjugates arepreferably used in a method for detecting an analyte in a sample bycontacting the sample with one or more analyte-specific bindingpartners.

The method for detecting an analyte is preferably carried out as animmunoassay. This means that at least one of the analyte-specificbinding partners is an immunological binding partner. In this method thesample which is presumed to contain the analyte is incubated with animmunologically specific binding partner. In the case of an antigen testfor example for tumour markers such as PSA, an antibody or a fragmentthereof which specifically binds to the analyte, i.e. the tumour antigenPSA, is the said immunologically specific binding partner. In methodsfor detecting antibodies to a certain antigen (e.g. anti-HCV antibodies)the corresponding antigen can for example be used as the immunologicallyspecific binding partner. The specific binding is detected by means ofthe inventive conjugate whose incorporated dye serves as a label. Thebiomolecules immobilized on the metal oxide particles act as specificbinding partners for the analyte or as specific binding partners for asubstance which in turn is specifically bound to the analyte.

For example in a diagnostic test procedure, streptavidin or avidin canbe conjugated as the biomolecule to the metal oxide particle. Theconjugate then binds to the biotin group of a molecule (for example apeptide antigen or a nucleic acid sequence) that is itself biotinylated.

Immunoassay procedures and nucleic acid test procedures are familiar toa person skilled in the art.

The conjugates according to the invention are preferably used in a testbased on a test strip. The following describes, as an example, how atest strip is constructed and how such a test procedure is carried out.

Test strips are usually composed of a carrier material on which anapplication fleece, a membrane and a suction fleece are mounted. Theconjugate according to the invention whose biomolecules are specific forthe analyte and optionally other specific binding partners for theanalyte are applied and dried upstream of the chromatography directioni.e. above the starting point for the sample liquid. The specificbinding partners and the inventive conjugate do not begin to migratechromatographically until contact with a liquid i.e. with the sample.Various proteins are also applied to the membrane in the direction ofchromatography in the form of two successive strips or lines.

An immobilized binding partner that is specific for the analyte islocated on the first line (result line). A molecule such as streptavidincan also be bound to the first line to which biotinylated,analyte-specific binding partners can then bind. In this case thebiotinylated, analyte-specific binding partners as well as the conjugatemust be applied above the starting point of the test strip andchromatographed together with the sample. A binding partner whichspecifically binds the biomolecules of the inventive conjugate isapplied to the second line in the direction of chromatography (controlline).

As the sample liquid migrates from the starting point of the test stripthrough the strip, the conjugate according to the invention andoptionally the analyte-specific binding partner also begin to migratetowards the liquid front. In this process the analyte from the samplespecifically binds to the binding partners immobilized on the firstline. The inventive conjugate also binds to the analyte to form asandwich that can be detected by means of the colour of the metal oxideparticles. The liquid in the test strip runs further up to the end ofthe test strip. In this process the inventive conjugate that is notconsumed by analyte binding is captured on the second line by thebinding partner that specifically binds the biomolecules of theconjugate. One can see on the basis of the colouration of the controlline that the chromatography in the test strip has basically workedand/or is completed.

Another subject matter of the invention is a diagnostic test stripwhich, in addition to the conjugate according to the invention, containsall other components necessary to carry out the chromatographic test.

According to the invention the conjugates can also be used in nucleicacid hybridization assays. In this case a nucleic acid probe whichspecifically hybridizes with a nucleic acid sequence to be detected iscoupled as a biomolecule with the metal oxide particles that arecoloured according to the invention. The nucleic acid sequence from thesample or from a mixture that is for example obtained by PCRamplification can be specifically detected by means of the dye containedin the metal oxide particles.

According to the invention the conjugates comprising the metal oxideparticles according to the invention and a biomolecule can also be usedin array or chip systems. Such systems are miniaturized test designs.Spatially separated reagent spots are applied with a very small spacingwhich is in the micrometer range to the surface of suitable solid phasessuch as plastics, glass, metals or metal oxides. These reagent spotscontain the specific binding particles required to carry out therespective detection method. Such detection methods enable numerousdifferent analytical parameters to be detected simultaneously andrapidly in a very small space using little material and sample. Theconjugates according to the invention comprising metal oxide particlescoloured with halogen-containing dyes and biomolecules can also be usedas detection reagents in these array or chip systems. Suitable dyes forcolouring the metal oxide particles are preferably fluorescent dyes andespecially those that enable a time-resolved measurement offluorescence. In particular the conjugates according to the inventionenable differently coloured and/or different conjugates loaded withdifferent biomolecules to be used in order to simultaneously detectdifferent analytes by means of the different (fluorescent) dyes. Sucharray systems have proven to be particularly advantageous for nucleicacid hybridization assays.

The simultaneous detection of a plurality of different analytes (forexample HIV- and HCV-specific nucleic acids in a sample or HIV- andHCV-specific antibodies in a sample) by means of the conjugatesaccording to the invention which are each differently coloured and/orloaded with different biomolecules is not limited to an application inarray systems but is particularly appropriate therefor.

All body fluids can be used as the sample material for all diagnostictest methods. Whole blood, serum, plasma, urine, sweat or saliva arepreferably used.

Another subject matter of the invention is the use of the conjugates ofmetal oxide particles according to the invention and biomolecules in adiagnostic, preferably immunological method to detect an analyte in asample.

A diagnostic reagent which contains conjugates according to theinvention is also a subject matter of the invention. The reagent canadditionally contain the buffer additives, salts or detergents known toa person skilled in the art.

A test kit which contains the conjugates according to the invention andother common reagents known to a person skilled in the art for carryingout a test is also one of the preferred embodiments of the presentinvention.

Many important insecticides have a high halogen content. Theseinsecticides are preferred target molecules for the process according tothe invention for non-covalent incorporation into metal oxide particlesand in particular into silicate particles.

The digestive tract of insects and especially of insect larvae differsfundamentally from that of mammals. Whereas there is a strongly acidicpH the stomach of mammals, food is digested in the digestive tract ofinsect larvae in a strongly alkaline pH range.

Metal oxide particles especially those based on silicate or mixed oxideparticles containing more than 20% silicate have the special propertythat they swell under alkaline pH conditions such as those that are forexample present in the digestive tract of insects and releasenon-covalently incorporated components, in particular polyhalogenatedinsecticides. Since the insecticide target molecules are not covalentlybound in the particles according to the invention, they are released andare effective in the insect intestine i.e. precisely at the intendedsite of action.

The inclusion of insecticide agents in metal oxide particles by thesol-gel process of the present invention additionally has the effectthat the agents are for example protected from water. The insecticidaleffect only occurs after intake of food by the insect. Sol-gel particlesaccording to the invention containing insecticides with incorporatedinsecticidal agents are less poisonous and/or more environmentallyfriendly than the free agents.

Halogen-containing dyes having spectral properties that are importantfor the printing industry can be incorporated into sol-gel particles inthe process according to the invention. Such particles are usedespecially as an admixture in so-called toners.

The halogen-containing substances which can be incorporated according tothe invention into metal oxide particles include many substances whichabsorb and suppress damaging UV light or release it again as longerwavelength less damaging light. Metal oxide particles according to theinvention which contain such substances are preferably used in thecosmetic industry especially as sunscreen agents.

The invention is further elucidated by the following examples,publications and figures the protective scope of which results from theclaims. The described processes are to be understood as examples whichstill describe the subject matter of the invention even aftermodifications.

DESCRIPTION OF THE FIGURES

FIG. 1: Schematic representation of homogeneous (type 1) orheterogeneous (core-shell (type2), currant cake (type 3) particleshaving an inorganic-oxidic matrix.

FIG. 2: Structural formula ofTris-[4,4,4-trifluoro-1-(2-naphthyl)-1,3-butanedione]-Eu(III) complex(Eu(NTA)3 complex).

FIG. 3: UV-Vis spectrum of the Eu(NTA)3 complex in CH₂Cl₂/EtOH (1:1)λ_(abs).=333 nm.

FIG. 4: fluorescence spectrum of the EU(NTA)3 complex in CH₂Cl₂/EtOH(1:1) λ_(exc).=333 nm.

Assignment of the Fluorescence Bands to the Spectral TransitionsEmission bands assignment* λ_(em) = 578 nm ⁵D₀ → ⁷F₀ λ_(em) = 590 nm ⁵D₀→ ⁷F₁ λ_(em) = 612 nm ⁵D₀ → ⁷F₂ λ_(em) = 651 nm ⁵D₀ → ⁷F₃ λ_(em) = 699nm ⁵D₀ → ⁷F₄*Lit.: R. Reisfeld et al., J. of alloys and Compounds 300-301 (2000),147-151

FIG. 5: Measurement of a solid specimen of silicate particles containingthe Eu(NTA)3 complex which were fixed on a microscope slide.

Settings:

-   power=950 mV, slit widths=EX/EM=10/1-   light source=xenon lamp-   λ_(exc).=333 nm-   λ_(em).=613 nm

FIG. 6: IR spectrum of the silicate particles doped with the Eu(NTA)3complex on a pressed piece of KBr

Assignment of the Bands

-   ν(O—H)=3430 cm⁻¹-   δ(H₂O)=1640 cm⁻¹-   ν(Si—O—Si)=1100 cm⁻¹ (as)-   ν(Si—O—Si)=800 cm⁻¹ (sym)-   δ(Si—O—Si)=471 cm⁻¹ (?)

Assignment according to: Fendler, J. H., Nanoparticles andnanostructured Films, Wiley-VCH 1998, 180-183.

FIG. 7: Raman spectrum of a solid specimen of silicate particles dopedwith an Eu(NTA)3 complex

Assignment of the Bands:

-   ν(C—H, aliph.)=2943, 2875 cm⁻¹ (as)-   δ(CH₃, CH₂)=1452 cm⁻¹-   ν(Si—O—Si)=1068 cm⁻¹ (as)-   ν(Si—O—Si)=839, 793 cm⁻¹ (sym)-   δ(Si—O—Si)=482 cm⁻¹

Assignment according to: J. H. Fendler, supra

FIG. 8: TEM pictures of 130-158 nm silicate particles doped with 4.9μmol Eu(NTA)3 complex per g SiO₂ at 6300-fold (FIG. 8 left) and63000-fold enlargement (FIG. 8 right)

FIG. 9: VACP/MAS 13C solid NMR spectrum of the Eu(NTA)3 complex

Interpretation:

-   129.4/126.7 ppm; arom. C—H-   61.2 ppm; CH₂—OH-   27.6 ppm; CH₂—CH₂—CF₃-   17.4 ppm; CH₃—CH₂—OH-   4.5 ppm; Si—CH₂—CH₂—CF₃

FIG. 10: MAS ²⁹Si solid NMR of silicate particles doped with theEu(NTA)3 complex.

Integration of the Signals Yielded the Following Distribution:

-   110.7 ppm; Q4-groups, 70.54%-   101.1 ppm: Q3-groups, 27.24%-   91.0 ppm: Q2-groups, 2.21%

ABBREVIATIONS USED

-   AMEO 3-aminopropyltriethoxysilane-   <Dig> anti-digoxigenin-   ETEO ethyltriethoxysilane-   GF20 2(3-triethoxysilylpropyl)-succinic anhydride-   GLYMO glycidoxypropyltrimethoxysilane-   Ig immunoglobulin-   LCR LightCycler Red-   MAB monoclonal antibody-   MEMO methacryloxypropyltrimethoxysilane-   MES 2(N-morpholino)ethanesulfonic acid-   MPTMO 3-mercaptopropyltrimethoxysilane-   BPLA bovine plasma albumin-   SA streptavidin-   Si—NP silicate nanoparticle-   TEOS tetraethoxysilane-   TMES tetramethoxysilane

EXAMPLE 1 General protocol for preparing lanthanide(III)-tris-4,4,4-trifluoro-(1-naphthoyl)-1,3-butanedione complexes

800 mg (3 mmol) 4,4,4-trifluoro-1-(2-naphthoyl)-1,3-butanedione wasdissolved in 15 ml ethanol. Subsequently 3 ml of a 1 M NaOH solution wasadded to this solution. 1 mmol lanthanum (III) chloride or lanthanum(III) nitrate was dissolved in 5 ml water in a dropping funnel and thenslowly added dropwise to the reaction solution. Afterwards a further 100ml water was added to the reaction mixture and it was stirred for 1 hourat 65° C. The product was filtered off as a pale yellow solid and washedthree times with 5 ml water and ethanol each time. It was finally driedfor 3 hours at 120° C. in a drying cabinet.

This general procedure was used to prepare terbium (III), gadolinium(III), dysprosium (III), and erbium (III) complexes.

EXAMPLE 2 Review of Physical Dye Incorporation into Fluorine-Free(“Normal”) Silicate Nanoparticles (Si—NP) and Organofluorine-Modified(Fluorinated) Si—NP 2.1 Preparation of Normal Silicate ParticlesColoured with LightCycler Red 640™ (Reference Particles)

41 mg (4.29*10⁻⁵ mol) LightCycler Red 640™ LCR 640 was dissolved in 330ml 99% ethanol. 168 ml demineralized water and 11 ml of a 14 molarammonium hydroxide solution were added to this solution. The solutionwas heated to 35° C. After a thermal equilibrium was established, 24 ml(107 mmol) tetraethoxysilane (TEOS) was added while stirring vigorously.The reaction was fully completed after 24 h. A coloured dispersionhaving a solids content of about 2% by weight was obtained. Theparticles have a size of about 135 nm diameter. These particles werepurified of non-incorporated dye by centrifuging and redispersing threetimes in fresh ethanol.

2.2 Preparation of Fluorinated Silicate Particles Coloured withLightCycler Red 640™

a) Protocol for Particles Containing 0.3% Fluoroalkylsilane

23.8 μmol LightCycler Red 640™, then 6 ml TEOS and 30 μl (155 μmol)3,3,3-trifluoropropyltrimethoxysilane (ratio of LCR 640™:fluoroalkylsilane=1:7) were added to a solution comprising 165 ml EtOH,84 ml H₂O and 5.5 ml NH₄OH heated to 35° C. After stirring for 5 minutesthe remaining 6 ml TEOS was added to the reaction mixture. The reactionwas terminated after 8 h and the particles were separated bycentrifugation. The particles were redispersed in H₂O and purified bycentrifuging and redispersing several times.

b) Protocol for Particles Containing 0.2% Fluoroalkylsilane

23.3 μmol LightCycler Red 640™, then 5 ml TEOS and 20 μL (119 μmol)3,3,3-trifluoropropyltrimethoxysilane (ratio of LCR 640™:fluoroalkylsilane=1:5) were added to a solution comprising 31 ml EtOH,20 ml H₂O and 7 ml NH₄OH heated to 30° C. After stirring for 5 minutesthe remaining 5 ml TEOS was added to the reaction mixture. The reactionwas terminated after 8 h and the particles were separated bycentrifugation. The particles were redispersed in H₂O and purified bycentrifuging and redispersing several times.

2.3 Preparation of Silicate Particles Doped withEu(III)-tris-4,4,4-trifluoro-1-(2-naphthyl)-1,3-butanedione

The Eu(III)-tris-4,4,4-trifluoro-1-(2-naphthyl)-1,3-butanedione complexwas prepared according to the instructions of Charles, R. G., andRoedel, E. P., J. Inorg. Nucl. Chem. 29 (1967) 715-723.

a) Simultaneous Addition of Alkoxide and a Mixture of PolyhalogenatedAlkylalkoxysilane and Halogen-Containing Target Molecule

20 ml TEOS and a mixture of 20 mgtris-[4,4,4-trifluoro-1-(2-naphthyl)-1,3-butane-dione]-Eu(III), 1 mldichloromethane and 0.25 ml 3,3,3-trifluoropropyltrimethoxysilane wereadded to a solution comprising 61 ml water, 40 ml ethanol and 14 mlammonium hydroxide solution heated to 30° C. The reaction mixture wasstirred for 4 hours at 30° C. and for a further 10 h at roomtemperature. The particles were purified by centrifugation and firstlyredispersed in ethanol and then in water in a subsequent washing step.

-   Incorporation rate (complex): 4.9 μmol/g SiO2-   Calculated Eu content: 0.07%-   Eu content found by X-ray fluorescence analysis (RFA): 0.05%-   Particle size from TEM: 130-158 nm    b) Successive Addition of Alkoxide and a Mixture of Polyhalogenated    Alkylalkoxysilane and Halogen-Containing Target Molecule

61 ml water and 40 ml ethanol were heated to 30° C. in a 250 ml roundbottomed flask. Then 14 ml ammonia solution and 10 ml TEOS were added.In parallel 19.4 mg (20.4 μmol)tris-[4,4,4-trifluoro-1-(2-naphthyl)-1,3-butanedione]-Eu(III) wasdissolved in 1 ml dichloromethane, and 250 μltrifluoropropyltrimethoxysilane was added to the solution in anultrasonic bath. After 5 minutes the solution was added dropwise to thepreparation and stirred for a further 5 minutes. Subsequently another 10ml TEOS was added and the reaction mixture was stirred for 4 hours at30° C. and for a further 10 hours at room temperature. It was purifiedin several washing cycles using ethanol and water.

2.4 Preparation of the erbium (III) tris-(2,2′-bipyridyl)trichloridecomplex

1.1 g (7 mmol) 2,2′ bipyridine and 250 mg (0.7 mmol) erbium (III)nitrate (as an undefined hydrate complex) were added to 60 ml methanol.The reaction mixture was heated for 2 h to 60° C. while stirringvigorously. The complex precipitated as a yellow powder on cooling.

Incorporation into silicate particles was carried out as described inexample 2.3a.

2.5 Preparation of the terbium (III) tris-(2,2′-bipyridyl)trichloridecomplex

1.47 g (9.43 mmol) 2,2′ bipyridine and 250 mg (9.43*10⁻⁴ mol) terbium(III) chloride hexahydrate complex were added to 60 ml methanol. Thereaction mixture was heated for 2 h to 60° C. while stirring vigorously.The complex precipitated as a yellow powder on cooling.

Incorporation into silicate particles was carried out as described inexample 2.3a.

2.6 Preparation of the terbium (III)tris-(1,10-phenanthroline)trichloride complex

482 mg (2.67 mmol) 1,10-phenanthroline and 250 mg (0.94 mmol) terbium(III) chloride hexahydrate were added to 20 ml methanol. The solutionwas stirred for 2 h at 60° C. and subsequently slowly cooled to roomtemperature (overnight). The yellow solution obtained in this manner wasoverlayered with n-pentane. The complex precipitates as a powder.

Incorporation into silicate particles was carried out as described inexample 2.3a.

2.7 Summary of the Incorporation Behaviour of Various Dyes intoUnmodified and Halogen-Modified Silicate Particles

incorporation into Incorporation of Normal Si—NP fluorinated Si—NPfluorine-free dyes ⊖ Tb(III)-bipy ⊖ Tb(III)-bipy ⊖ Er(III)-bipy ⊖Tb(III)-phen ⊖ Tb(III)-phen Fluorinated or halo- ⊖ Eu(NTA)₃ ⊕ Eu(NTA)₃genated dyes ⊖ LCR 640 ⊕ Er(NTA)₃ ⊕ LCR 640Legend:⊖ incorporation negative,⊕ incorporation positivebipy = α,α′-bispyridinephen = 1,10-phenanthroline

EXAMPLE 3 Surface Modification with GF20 (Protocol for IntroducingCarboxyl Groups)

The dispersion obtained in example 2.1 and 2.2 should not exceed a pH of9.0. If necessary additional washing cycles have to be carried out(centrifugation/redispersion). 210 μl (75.4*10⁻⁵ mol)2-(3-triethoxysilylpropyl)-succinic anhydride (GF20) was added to theresulting ethanolic dispersion in a volume of 250 ml while stirringvigorously. The reaction solution was stirred for 15 h at 40° C. Theparticles were purified by centrifugation and redispersion in water.This purification step was repeated a further two times. An aqueousdispersion of surface-modified particles is obtained with a coveragedensity of ca. 2 CO₂H groups/nm² particle surface.

EXAMPLE 4 Preparation of Conjugates of Silicate Particles Coloured withLightCycler Red 640 and Anti-Digoxigenin Antibodies (<Dig> Conjugates)

10 mg silicate particles (0.5 ml 2% suspension) was centrifuged for 30min at 15000 rpm. The supernatant was removed and the pellet wasresuspended in 1 ml 2 mM MES buffer pH 6.5. This washing process wasrepeated once more. Subsequently 100 μl 100 mM MES buffer pH 6.5, 100 μl2% (w/v) sulfo-N-hydroxy-succinimide (S—NHS; Pierce No. 24510) in MESbuffer pH 6.5 and 100 μl 0.2% (w/v)1-ethyl-3-(3-diaminopropyl)-carbodiimide hydrochloride (EDC; Pierce No.22980ZZ) in MES buffer pH 6.5 was added. After 20 min incubation periodon a roller incubator, it was centrifuged for 30 min at 15000 rpm andthe supernatant was taken off. The pellet was redispersed in 867 μl 2 mMMES buffer pH 6.5, and 133 μl MAB<Dig>M-IgG solution (monoclonalanti-digoxigenin IgG antibody from the mouse; concentration=15 mg/ml)was added. Afterwards it was incubated for 2 h at room temperature (RT).Subsequently 1 ml of a 2% solution of bovine plasma albumin (RPLA) in 5mM potassium phosphate buffer pH 7.4 was added and it was incubated fora further 60 min at RT. The particle conjugates were centrifuged, thesupernatant was removed and the pellet was resuspended in 1 ml 5 mMbuffer. The potassium phosphate washing process was repeated twice andthe particles were resuspended in 0.5 ml 2% RPLA in 5 mM Hepes buffer pH7.4 after the last centrifugation step.

EXAMPLE 5 Use of <Dig> Silicate Particle Conjugates in a Strip Test

The test strips required to carry out the experiments consist of aplastic foil onto which an application fleece, a membrane and a suctionfleece are glued. Two proteins, streptavidin and anti-mouse-IgGantibody, are immobilized on different lines on the membrane.

Poly-SA was immobilized on the target or result line i.e. the first linein the direction of chromatography and should specifically captureparticles bound to digoxigenylated and biotinylated peptide by means ofthe biotin binding. The anti-mouse IgG antibodies were immobilized onthe control line i.e. the second line in the direction ofchromatography. These anti-mouse IgG antibodies should capture allexcess particles that were not bound on the result line (conjugates ofanti-digoxigenin antibodies from the mouse and the silicate particles).

Depending on the test strip variant the application fleece wasimpregnated with the sample material to be examined i.e. with 1 μg/ml or0 μg/ml of a biotinylated and digoxigenylated peptide. A 100 mM Hepesbuffer pH 7.5 (50 mM NaCl, 70 mM urea, 1 mM EDTA, 2% BPLA) was used todilute the silicate particles and rewash the test strips. The silicateparticles were diluted in Hepes buffer to a final concentration of 100μg/ml.

Subsequently 60 μl of the described silicate particle dilution waspipetted on the reagent fleece and chromatographed for 10 min.Afterwards 40 μl Hepes buffer was pipetted onto the reagent fleece andchromatographed for a further 10 min. Finally the test strip wasevaluated. Only the control line was visible in the absence of thepeptide (=analyte) and in the presence of the peptide the result linewas additionally visible.

The conjugates according to the invention of silicate particles colouredwith halogen-containing dyes and biomolecules (in this caseanti-digoxigenin antibodies) are thus suitable as detection reagents inan immunological test strip.

LIST OF REFERENCES

-   Badley, R. D., et al., Langmuir 6 (1990) 792-801-   Byers, C. H., et al., Ind. Eng. Chem. Res. 26 (1987) 1916-1923-   Charles, R. G., und Roedel, E. P., J. Inorg. Nucl. Chem. 29 (1967)    715-723-   EP 1 036 763-   Fendler, J. H., Nanoparticles and Nanostructured Films, Wiley-VCH    1998, 180-183-   Giesche, H., and Matijevic, E., Dyes and Pigments 17 (1991) 323-340-   Giesche, H., J. European Ceramic Soc. 14 (1994) 189-204-   Harris, T., et al., J. Non-Cryst. Solids 121 (1990) 307-403-   Horn, D., and Rieger, J., Angew. Chem. 113 (2001) 4460-4492-   Kawaguchi, H., Prog. Polym. Sci. 25 (2000) 1171-1210-   Kron, J., et al., 2. Wörlitzer Workshop, Tagungsband 2000-   Matijevic, et al., Dyes and Pigments 17 (1991) 323-340-   Matsoukas, T., and Goulari, E., Colloid J. Interface Sci. 124 (1988)    252-261-   Matsoukas, T., and Gulari, E., Colloid J. Interface Sci. 132 (1989)    13-21-   Philipse, A. P., and Vrij, A., Colloid J. Interf. Sci. 12 (1989)    121-136-   Quellet, et al., Colloid J. Interface Sci. 159 (1993) 150-7-   Reisfeld, R., et al., J. of Alloys and Compounds 300-301 (2000),    147-151-   Schwert, R., Dissertation, Würzburg (?)2000-   Shibata, S., et al., J. Sol-Gel Sci. and Techn. 10 (1997) 263-268-   Stöber, et al., Colloid J. Interface Sci. 26 (1968) 62-69-   U.S. Pat. No. 5,102,763-   U.S. Pat. No. 5,209,998-   Van Blaaderen, A., and Vrij, A., Adv. Chem. Ser. 234 (1994) 83-111-   Van Blaaderen, A., and Vrij, A., Colloid J. Interface Sci.,    156 (1993) 1-18-   Van Blaaderen, et al., Langmuir 8 (1992) 2921-2931-   Van Helden, A. K., and Vrij, A., Colloid J. Interf. Sci. 81 (1981)    354-368-   Verhaegh, and Van Blaaderen, A., Langmuir 10 (1994) 1427-1438-   WO 93/10190

1. Sol-gel process for producing a metal oxide particle which containsat least one target molecule containing halogen in which, starting fromknown metal oxide precursors, the said precursor and the said targetmolecule are used, characterized in that a polyhalogenated metalalkylalkoxy compound is additionally used in the said sol-gel process.2. Sol-gel process for producing a metal oxide particle containing atleast one halogen-containing target molecule as claimed in claim 1,comprising the steps a) production of a mixture containing the targetmolecule and a polyhalogenated metal alkylalkoxy compound, b) startingthe sol-gel process with the metal oxide precursor, c) adding thesolution from a), d) optionally further addition of the metal oxideprecursor and e) ending the sol-gel process.
 3. Process as claimed inclaim 2, characterized in that 90 to 10% of the metal alkoxide precursoris used in step a) and 10 to 90% of the metal oxide precursor is used instep d).
 4. Process as claimed in one of the claims 1 to 3,characterized in that based on the initial amount of metal oxideprecursor between 0.04 and 0.4 mol % polyhalogenated metal alkylalkoxycompound is used.
 5. Process as claimed in one of the claims 1 to 4,characterized in that based on the initial amount of metal oxideprecursor between 0.1 and 10% by weight target molecule is used. 6.Process as claimed in one of the claims 1 to 5, characterized in thatthe halogen-containing target molecule is chlorinated or fluorinated. 7.Process as claimed in one of the claims 1 to 6, characterized in thatthe metal oxide is composed of B₂O₃, Al₂O₃, SiO₂, ZrO₂ or TiO₂ or mixedoxides thereof.
 8. Metal oxide particle obtainable by the sol-gelprocess as claimed in one of the claims 1 to
 7. 9. Use of a particle asclaimed in claim 8 as a label for biomolecules.
 10. Use of a particle asclaimed in claim 8 as a sunscreen agent.
 11. Use of a particle asclaimed in claim 8 as a toner.
 12. Use of a particle as claimed in claim8 as an insecticide.